Water-soluble polymers such as polyethylene glycol, when bound to biomolecules as typified by protein drugs, are known to confer clinical usefulness in ways that bring about effects such as improved physical and thermal stability, resistance to protease, and solubility as well as decreased in vivo distribution volume and improved retention in blood (see Inada et al., J. Bioact and Compatible Polymers 5,343(1990); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9,249 (1992); and Katre, Advanced Drug Delivery Reviews 10,91(1993)).
A variety of methods are available for binding natural interferon-β or interferon-β having a primary structure identical to natural one to a water-soluble polymer polyethylene glycol (PEG). For example, Katre et al have applied the amino group modification of lysine or the like to the PEGylation of interferon-β (see U.S. Pat. Nos. 4,766,106 and 4,917,888 and International Publication No. WO87/00056). Specifically, they have reported a conjugate obtained by binding a water-soluble polymer (PEG) having a molecular weight of 300 to 100,000 to recombinant interferon-β or IL-2 via 1 to 10 lysine residues in the amino acid sequence thereof. Alternatively, a technique for binding PEG to an amino group in lymphokine has already been reported in “Chemically modified lymphokine and production thereof” (see JP Patent Publication (Kokai) No. 60-226821A (1985)). However, in reality, interferon-β bound with PEG by these methods has interferon-β activity decreased to less than 10% and can not be in practical use.
No previous report has described a technique for selectively binding PEG to the amino group of particular lysine in interferon-β. If it is possible to select and specifically modify lysine that minimizes the rate of reduction in interferon-β biological activity caused by PEG binding, reduction in the total amount of proteins administered as a pharmaceutical drug leads to fewer side effects to patients and further to easier quality control.
On the other hand, a method is also known which uses reductive alkylation without involving lysine residues to selectively bind a water-soluble polymer to the amino terminus of interferon through reaction at pH suitable for the selective activation of the amino-terminal α-amino group of the interferon (see JP Patent Publication (Kokai) No. 9-25298A (1997)). However, in reality, the PEGylation of interferon-β by this method does not give mono-PEGylation and brings about nonselective PEGylation at any lysine residue or the N terminus, resulting in the generation of a heterogeneous mixture without sufficient antiviral activity and cell growth-inhibiting activity.
More importantly, purified interferon-β N-terminally bound with PEG is also known to have remaining activity (ratio with respect to interferon-β activity before binding) dramatically decreased when the PEG has a molecular weight higher than 20,000 and to completely lack activity when the PEG has a molecular weight of 40,000, as reported by Pepinsky et al (see Pepinsky et al., The Journal of Pharmacology and Experimental Therapeutics, vol. 297, p 1059-1066, (2001)).
As for interferon-α, Bailon et al have produced interferon-α nonselectively mono-PEGylated at the lysine residue with a branched polymer PEG having a molecular weight of 40,000 (Bailon et al., Bioconjugate Chem. 12, 195 (2001)). However, they have reported that the remaining activity of interferon-α bound with PEG having a molecular weight as high as 40,000 is significantly decreased, as in the case with interferon-β N-terminally bound with PEG, and is 7%.
Namely, it is difficult to directly apply techniques (the number and position of PEG bound) that have been developed for modification with low molecular weight PEG to high molecular weight PEG. Thus, a novel technique has been required for producing a highly active interferon-β complex bound with PEG having a molecular weight (20,000 or higher) necessary to sufficiently obtain effects such as extended in vivo circulatory half-life and decreased clearance values that lead to usefulness as a pharmaceutical drug.
As described above, there has been no report so far on the selection of a lysine residue to be modified for avoiding reduction in the activity of interferon-β bound with a high molecular weight water-soluble polymer, and on a technique for this purpose. Moreover, there has been no report that the selective binding of a high molecular weight water-soluble polymer such as PEG to any one of 11 lysine residues present in interferon-β produces a highly active interferon-β complex.